As described in the previous annual report, we had been unable to purify the recombinant preS1 peptide or a fusion protein containing it mainly because of its low level of expression. We have since prepared a new construct which abundantly expresses a fusion protein (ca. 80,000 daltons) in inclusion bodies in Escherichia coli. After solubilization with urea and removal of phospholipids with 1,1,2trichlorotrifluoroethane, the crude fusion protein solution was treated with human Factor Xa to release the preS sequence. However, a 10,000 dalton peptide consisting of the amino-terminal 91 amino acids of preS1 was isolated and purified (by Mono-Q chromatography). This was the main product, rather than the expected 16,000 dalton peptide consisting of the inserted preS1-preS2 sequence. Factor Xa cleaves the peptide bond that involves the carboxyl group of arginine, not only at the inserted tetrapeptide site, -Ile-Glu-Gly-Arg-, but also at -Gly-Arg- which is present at positions 90 and 91 of preS1. This recombinant preS1 peptide was labeled by conjugating with I-125 Bolton-Hunter (BH) reagent. The labeled BH-preS1 peptide appeared to behave immunologically as unlabeled preS1 when RIAs were performed with either commercial monoclonal preS antibodies or our own specific polyclonal rabbit anti-synthetic preS1 peptide (21-47). Human hepatocyte membranes were prepared from autopsied liver specimens (kindly provided by the NIH Clinical Center and D.C. General Hospital). The labeled preSl can bind to human hepatocyte membranes and the binding can be inhibited by either the unlabeled ligand or anti-preS1 monoclonal antibodies. Two rabbits have been immunized with this peptide, but anti-preS1 titers remain low after several booster doses. Work is still in progress.